Characterization of a nuclear matrix-associated endonuclease

The nuclear matrix is a proteinaceous network that serves as a structural framework of the eukaryotic nucleus. It was isolated by treating purified nuclei with nucleases, followed by extractions with high-salt and non ionic detergent (B ere z n e y and C 0 f fey 1974). Aside from its welldefined structural role, the nuclear matrix is involved in many nuclear functions. It participates in chromatin organization and every aspect of DNA metabolism: from DNA synthesis, gene transcription to DNA degradation (B ere z n e y et at. 1995; Get zen b erg 1994;G rom 0 v a et at. 1995). DNA degradation during apoptosis starts with the release of high-molecular weight DNA fragments (50-300 kb) from their points of attachment on the nuclear matrix structure. Although the nuclease that catalyses this process is still unidentified, the biochemical requirements of initial apoptotic DNA degradation are well established (S u nand C 0 hen 1994; K 0 k i I e v a 1995). Recently, it was shown that nonapoptotic cells contain a constitutive high-molecular weight fragmentation activity that is localized on the isolated nuclear matrix (S 0 I 0 v y a n et at. 2002). In this paper we describe for the first time an endonuclease that is associated with the rat hepatocyte nuclear matrix. At present, a role for such a nuclear matrix-associated enzymatic activity is unclear. One possibility is that the nuclear matrix-associated endonuclease is involved in apoptotic DNA degradation. In order to verify this assumption, the biochemical requirements of the nuclear matrixassociated nuclease were examined. Nuclear matrices were isolated from purified hepatocyte nuclei essentially as described previously (P 0 z nan 0 vic et at. 1996) except that the nuclei were subjected to endogenous nuclease digestion. Endonuclease activities in the protein fractions were established by activity-gel analysis. The method is based on the electrophoretic separation of proteins in 10% SDS-polyacrylamide gels containing 100 ~/mL salmon sperm DNA (R a u c h et at. 1997). After electrophoresis the gels were washed in renaturation buffer (50 mM Tris pH 7.0, 1 mM DTT) overnight at 4°C, and incubated for 24 h in the same buffer with the addition of different cations in order to activate nuclease activity/-ies. The gels were then stained with ethidium bromide and nuclease activities were detected as dark areas on a fluorescent background after transillumination of the gels with UV light. Using this method we examined the presence of endonucleases in rat hepatocyte nuclei and their redistribution in protein fractions released during the isolation of the nuclear matrix (Fig. lB). Initially, activity-gel analysis was performed at